(i) Field of the Invention
The present invention relates to plant cells and plants having an increased content of N-acetylated glucosamine derivatives. Furthermore, the present invention relates to plant cells and plants synthesizing glucosaminoglycans. The present invention also provides processes for producing said plants and compositions comprising said plant cells.
(ii) Description of the Related Art
The amino sugar glucosamine, glucosamine derivatives and polymers comprising glucosamine derivatives are used, inter alia, as food supplements for the prophylaxis of joint disorders in animals and man. In the medical field, too, some glucosamine derivative-containing polymers are used for treating disorders.
WO 06 032538 describes transgenic plants which had been transformed with nucleic acid molecules coding for hyaluronan synthases. The synthesis of hyaluronan in the plants in question could be demonstrated unambiguously.
WO 98 35047 (U.S. Pat. No. 6,444,878) describes a metabolic path for the synthesis of GlcNAc in plant cells where glucosamine is converted by a number of successive enzymatically catalyzed reaction steps with formation of the metabolites GlcNAc, N-acetylglucosamine 6-phosphate, N-acetylglucosamine 1-phosphate into UDP-GlcNAc. A metabolic path which was described as an alternative for plants comprises the conversion of fructose 6-phosphate and glutamine into glucosamine 6-phosphate which is then converted by a number of successive enzymatically catalyzed reaction steps with formation of the metabolites glucosamine 1-phosphate and N-acetylglucosamine 1-phosphate into UDP-GlcNAc. The conversion of fructose 6-phosphate and glutamine into glucosamine 6-phosphate is catalyzed by a protein having the activity of a glutamine:fructose 6-phosphate amidotransferase (GFAT) (Mayer et al., 1968, Plant Physiol. 43, 1097-1107). Relatively high concentrations of glucosamine 6-phosphate are toxic for plant cells (WO 98 35047).
WO 00 11192 describes the endosperm-specific overexpression of a nucleic acid molecule from corn coding for a protein having the enzymatic activity of a plant GFAT in transgenic corn plants with the aim of synthesizing a cationic starch having 2-amidoanhydroglucose molecules in plants. The metabolic path described which, according to the description of WO 00 11192, should result in the incorporation of 2-aminoanhydroglucose into the starch, comprises inter alia the incorporation of UDP-glucosamine by starch and/or glycogen synthases into the starch. It was possible to demonstrate increased amounts of UDP-glucosamine in the flour of endosperm of the transgenic corn plants in question overexpressing a nucleic acid molecule coding for a protein having the enzymatic activity of a plant GFAT translationally fused with a plastid signal peptide. When the protein having the enzymatic activity of a GFAT was expressed without signal peptide, it was possible to demonstrate an increased amount of glucosamine 1-phosphate in the corresponding flour from corn endosperm tissue. It was not possible to detect cationic starch or increased amounts of N-acetylated glucosamine derivatives, such as, for example, UDP-GlcNAc or N-acetylglucosamine 6-phosphate, in the transgenic plants.
The amino sugar beta-D-glucosamine (glucosamine) and/or derivatives of glucosamine are components of various polymers (glucosaminoglycans) which, inter alia, are essential components of the exoskeleton of arthropods, the extracellular matrix of mammals or the exopolysaccharides of some bacterial microorganisms.
Thus, for example, N-acetyl-D-glucos-2-amine (N-acetylglucosamine, GlcNAc) is a glucosamine derivative acetylated at the nitrogen atom. GlcNAc is, for example, a molecular building block of hyaluronan (beta-1,4-[glucuronic acid beta-1,3-GlcNAc]n), which is an essential component of the synovial fluid.
In the medical field, hyaluronan-containing products are currently used for the intra-articular treatment of arthrosis and as ophthalmics used for eye surgery. Derivatized cross-linked hyaluronan is used for treating joint disorders (Fong Chong et al., 2005, Appl Microbiol Biotechnol 66, 341-351). In addition, hyaluronan is a component of some rhinologics which, for example in the form of eye drops and nasalia, serve to moisten dry mucous membranes. Hyaluronan-containing solutions for injection are used as analgesics and antirheumatics. Patches comprising hyaluronan or derivatized hyaluronan are employed in wound healing. As dermatics, hyaluronan-containing gel implants are used for correcting skin deformations in plastic surgery. In cosmetic surgery, hyaluronan preparations are among the suitable skin filler materials. By injecting hyaluronan, for a limited period of time, it is possible to smooth wrinkles or to increase the volume of lips.
In cosmetic products, in particular in skin creams and lotions, hyalauronan is frequently used as a moisturizer by virtue of its high water-binding capacity. Furthermore, hyaluronan-containing preparations are sold as so-called neutraceuticals (food supplements) which can also be used in animals (for example dogs, horses) for the prophylaxis and alleviation of arthrosis.
The catalysis of the hyaluronan synthesis is effected by a single membrane-integrated or membrane-associated enzyme, i.e. hyaluronan synthase (DeAngelis, 1999, CMLS, Cellular and Molecular Life Sciences 56, 670-682). Hyaluronan synthase catalyzes the synthesis of hyaluronan from the substrates UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylglucosamine (UDP-GlcNAc).
Hyaluronan used for commercial purposes is currently isolated from animal tissues (roostercombs) or prepared fermentatively using bacterial cultures.
Proteoglycans, a class of glycoproteins, are, inter alia, an essential component of cartilage and have, attached to a core protein, glucosaminoglycans composed of repetitive disaccharide units. The repetitive disaccharide units for their part are covalently attached to the core protein via a characteristic carbohydrate binding sequence. Depending on the composition of the disaccharide units, a distinction is made, inter alia, of the glucosaminoglycans heparan/heparin sulfate, keratan sulfate and chondroitin/dermatan sulfate, whose disaccharide units each contain a molecule which is either glucosamine or a glucosamine derivative. In these substances, sulfate groups are introduced at various atoms or substituents of the disaccharide units, so that the respective substances mentioned are not uniform polymers but polymer groups summarized under the respective generic term. Here, the individual molecules of the polymer groups in question may differ both in the degree of sulfation and in the position of the monomers containing sulfate groups.
The synthesis of the disaccharide chain of the chondroitin/dermatan ([beta-1,4]-[glucuronic acid beta-1,4-N-acetylgalactosamine]n) is catalyzed by a chondroitin synthase starting with UDP-GlcA and UDP-N-acetylgalactosamine, an epimer of UDP-GlcNAc (Kitagawa et al., 2001, J Biol Chem 276(42), 38721-38726). The glucuronic acid molecules of chondroitin can be converted by an epimerase into iduronic acid. If more than 10% of the glucuronic acid molecules are present as iduronic acid, the polymer is referred to as dermatan. The introduction of the sulfate groups in various positions of the disaccharide chain of the chondroitin or the dermatan is then catalyzed by further enzymes, resulting in chondroitin/dermatan sulfate. Here, the degree of sulfation may differ from molecule to molecule.
For some time, chondroitin sulfate has been considered as a potential active compound for treatment of osteoarthritis (Clegg et al., 2006, The New England Journal of Medicine 354(8), 795-808).
The synthesis of the disaccharide chain of heparin/heparan (heparosan) ([alpha-1,4]-[glucuronic acid beta-1,4-glucosamine]n or [alpha-1,4]-[iduronic acid alpha-1,4-glucosamine]n) is catalyzed by a heparin/heparosan synthase from UDP-GlcA and UDP-GlcNAc (DeAngelis und White, 2004, J. Bacteriology 186(24), 8529-8532). The glucuronic acid molecules of the heparin/heparosan can be converted by an epimerase into iduronic acid. The introduction of the sulfate groups in various positions of the disaccharide chain of the heparosan is then catalyzed by further enzymes, giving rise to heparin sulfate or heparan sulfate. Heparin sulfate has a considerably higher substitution by sulfate groups than heparan sulfate. Heparin sulfate has about 90% iduronic acid molecules, whereas in the case of heparan sulfate the fraction of glucuronic acid molecules predominates (Gallagher et al., 1992, Int. J. Biochem 24, 553-560). As in the case of chondroitin/dermatan sulfate, in the case of heparin/heparan sulfate, too, the degree of sulfation may differ from molecule to molecule.
Heparin sulfate is used, inter alia, as an anticoagulant, for example for the prophylaxis and treatment of thromboses.
Chondroitin/dermatan sulfate and heparin/heparan sulfate are currently produced by isolation from animal tissues. Chondroitin sulfate is mainly isolated from bovine or shark cartilage, and heparin/heparan sulfate is isolated from porcine intestine or bovine lungs. Since the disaccharide chains of chondroitin/dermatan sulfate or heparin/heparan sulfate have no uniform sulfation pattern, it is difficult to obtain a uniform specific product. Accordingly, the products are always mixtures of molecules with varying degrees of sulfation.
The glucosaminoglycan chitin ([beta-1,4-GlcNAc]n) is one of the main components of the cell wall of fungi and the exoskeleton of insects, millipedes, arachnids and crustaceans and is a polymer which is insoluble in water. The enzyme chitin synthase catalyzes the synthesis of chitin by linking UDP-GlcNAc (Merzendorfer and Zimoch, 2003, J. Experimental Biology 206, 4393-4412).
As a raw material source for isolating chitin, use is to date mainly made of crustaceans (prawns, crabs) and fungi, such as, for example, Aspergillus spec., Penicillium spec. Mucor spec. WO 03 031435 describes, for example, a method for preparing GlcNAc by fermentation of yeasts. Depending on the method by which chitin is isolated from the raw material source in question, chitin contains in addition to GlcNAc also its deacetylated form glucosamine as a building block. If more than 50% of the building blocks are GlcNAc, the polymer is referred to as chitin, whereas polymers comprising more than 50% of glucosamine are referred to as chitosan. These days, glucosamine or derivatives thereof, such as, for example, GlcNAc, are produced by degradation of chitin. Chitin may either be deacetylated first, resulting in the formation of chitosan, or be degraded directly, resulting in the formation of GlcNAc.
Chitin can be deacetylated enzymatically with the aid of chitin deacetylases (Kafetzopoulos et al., 1993, Pro. Natl. Acad. Sci. 90, 2564-2568) or by chemical deacetylation.
The degradation of chitin or of chitosan can also take place both enzymatically (for example using chitinases, glucanases, beta-N-acetylglucosaminidases), and by chemical hydrolysis.
The degradation of chitosan or the deacetylation of GlcNAc results in the formation of glucosamine.
A substantial disadvantage of all methods for preparing amino sugars by degradation of chitin consists in the fact that, owing to incomplete hydrolysis and/or incomplete deacetylation, what is obtained is not a uniform product but a mixture of various mono- and oligomers.
An alternative process for preparing glucosamine with the aid of recombinant microorganisms, in particular Escherichia coli, which does not require the degradation of chitin, is described in US 2002/0160459.
For some time, glucosamine and glucosamine-containing substances, too, have been considered as potential active compounds for the treatment of osteoarthritis (Clegg et al., 2006, The New England Journal of Medicine 354(8), 795-808). Glucosamine or glucosamine-containing substances are also present in many food supplements. Foods enriched with GlcNAc are described, for example, in US 2006/0003965.
As already described, glucosaminoglycans, such as, for example chondroitin sulfate, heparin/heparan sulfate or chitin are currently isolated from animal tissues. In addition to the substances desired in each case, these tissues also contain other glucosaminoglycans. The separation of the individual glucosaminoglycans, if a complete separation is possible at all, is difficult and complicated. Furthermore, the potential presence, in animal tissues, of pathogenic microorganisms and/or of other substances, such as, for example, the BSE pathogen or the bird flu pathogen, which may cause diseases in man, represent a problem when using glucosaminoglycans isolated from animal tissue. The use of medicinal preparations contaminated with animal proteins may, in the patient, result in unwanted immunological reactions of the body (for hyaluronan preparations, see, for example, U.S. Pat. No. 4,141,973), in particular if the patient is allergic to animal proteins.
A further problem during the isolation of glucosaminoglycans from animal tissues consists in the fact that the molecular weight of the glucosaminoglycans is frequently reduced during purification, since animal tissues also contain enzymes which degrade glucosaminoglycan.
Glucosamine or derivatives thereof isolated from crustaceans frequently contain substances (proteins) which may trigger an allergic reaction in man. Glucosamine or derivatives obtained from fungi may contain mycotoxins.
The amounts (yields) of glucosaminoglycans which can be obtained in satisfactory quality and purity from animal tissues are low (for example hyaluronan from roostercombs: 0.079% w/w, EP 0144019, U.S. Pat. No. 4,782,046), which means that large amounts of animal tissues have to be processed.
The production of glucosaminoglycans with the aid of fermentation of bacteria is associated with high costs, since the bacteria have to be fermented in sealed sterile containers under complicated controlled cultivation conditions (for hyaluronan, see, for example, U.S. Pat. No. 4,897,349). Furthermore, the amount of glucosaminoglycans which can be produced by fermentation of bacteria strains is limited by the existing production facilities. Here, it has also been taken into account that, owing to physical limitations, it is not possible to construct fermenters for relatively large culture volumes. In this context, mention may be made in particular of homogeneous mixing, required for efficient production, of fed-in substances (for example essential nutrient sources for bacteria, reagents for regulating the pH, oxygen) with the culture medium, which, if at all, can be ensured in large fermenters only with high technical expenditure.
Furthermore, substances prepared from animal raw materials are unacceptable for certain ways of life, such as, for example, veganism or for kosher food preparation.
Plants do not naturally produce glucosaminoglycans, such as, for example, hyaluronan, chitin, heparan/heparin sulfate, keratan sulfate or chondroitin/dermatan sulfate.
For the synthesis of glucosaminoglycans, it is necessary, inter alia, for sufficient amounts of acetylated glucosamine derivatives (in particular UDP-GlcNAc) and/or UDP-GlcA to be available as substrate for the respective enzymes involved in the synthesis. There is no information with regard to the amounts of N-acetylated glucosamines present in plant cells. WO 2005 035710 describes a process which allows the glucosamine content of plant material to be increased by drying. The highest glucosamine content in fresh, wet plant material was determined for chicory with 10 mg of glucosamine per 1 kg of fresh weight, which, at a molecular weight of 178 for glucosamine, corresponds to about 56 nmol of glucosamine per 1 gram fresh weight of plant material. WO 2005 035710 contains no information concerning the content of N-acetylated glucosamine derivatives in plants.
Furthermore, from the prior art described above, it is evident that the paths of glucosamine metabolism in plants have not yet been fully elucidated. In WO 00 11192, it was possible to generate plants by transformation with a nucleic acid molecule coding for a protein having the activity of a plant GFAT, which plants had an elevated content of glucosamine derivatives (UDP-glucosamine or glucosamine 1-phosphate); however, increased amounts of N-acetylated glucosamine derivatives were not found.